When contamination goes undetected, it isn’t just the batch that’s at risk; it’s the patient. This is because the presence of microorganisms can reduce a drug’s efficacy and cause serious infections or other complications.
In pharmaceutical manufacturing, ensuring sterility requires a comprehensive strategy that spans the entire production lifecycle. This includes processing, environmental and personnel monitoring, and adherence to good manufacturing practices. Within this framework, sterility testing serves as the final quality control step, confirming that products designated as sterile are free from viable microorganisms and ensuring that contaminated products don’t reach patients.
To uphold the safety of pharmaceutical products, global regulatory bodies, including the US Pharmacopeia (USP), the European Pharmacopoeia (Ph. Eur.), and the Japanese Pharmacopoeia (JP), have established detailed requirements for sterility testing.
The following article will explore these requirements in greater detail, along with the best practices that pharmaceutical laboratories can implement to uphold sterility and compliance.
Regulatory requirements for sterility testing
To protect patient safety and ensure consistent product quality, pharmaceutical manufacturers must follow standardized sterility testing protocols as defined by various regulatory bodies. While regional differences exist, the core expectations are closely aligned. Regulations outline testing procedures, set requirements for the manufacturing environment, and emphasize the importance of aseptic conditions throughout production.
In the United States, sterility testing is governed by USP <71>, which outlines two compendial methods for detecting viable microorganisms: direct inoculation and membrane filtration. USP <71> also mandates method suitability testing, in which the chosen method is validated against the product to ensure that antimicrobial activity or product-specific properties do not interfere with microbial detection. This test must be carried out for new products and whenever experimental conditions change.1
USP <71> has been harmonized with Ph. Eur. 2.6.1 and JP 4.06 by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). As a result, official pharmacopoeial texts can be used interchangeably across ICH regions, promoting a unified approach to sterility testing.2
The FDA’s Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing complements USP <71> by offering a broader perspective on sterility assurance. It addresses air quality and filtration, personnel training, process validation, environmental monitoring, and sterility testing. In terms of sterility testing, it provides recommendations for controlling the testing environment, understanding test limitations, and investigating positive test results.3
For sterile medicinal products manufactured or distributed in the EU or UK, additional requirements are outlined in EU GMP Annex 1: Manufacture of Sterile Medicinal Products. This document gives direction on the design and control of facilities, equipment, systems, and procedures to prevent contamination in the final product. Annex 1 specifies that tests must be performed under aseptic conditions and on samples representative of the whole batch, including material most susceptible to contamination. It also emphasizes that sterility testing is only one of several measures for assuring sterility, and that it cannot be used to assure the sterility of a product that hasn’t met its design, procedural, or validation parameters.4
Sterility testing methods
The compendial methods for sterility testing are membrane filtration and direct inoculation, with the choice depending on product characteristics.
Membrane filtration is preferred for testing larger volumes or products containing particulate matter. In this method, the sample is filtered through a sterile 0.45 micron membrane that captures microorganisms. The filter is then transferred to suitable growth media—typically fluid thioglycollate medium (FTM) for anaerobic bacteria and tryptic soy broth (TSB) for fungi and aerobic bacteria—and incubated for a minimum of 14 days. This method offers increased sensitivity since the entire volume of the product is tested. However, it is resource-intensive and time-consuming, requiring multiple handling steps and specialized equipment. There is also the risk of membrane clogging with highly viscous or particulate-rich samples.
Direct inoculation is used for small-volume samples where filtration is impractical, such as with viscous or non-filterable products. In this method, the product is inoculated into both FTM and TSB growth media and incubated for a minimum of 14 days. While simpler and less resource-intensive, it is limited by the smaller sample volume. Additionally, because the product remains in the media throughout incubation, it may inhibit microbial growth or interfere with turbidity readings, making method suitability testing essential.
Regardless of the method, sterility test results are qualitative: either microbial growth is observed, or it is not. If no turbidity is present after the full incubation period, the batch can be reported as sterile. If any signs of growth appear, the test is considered positive, and additional tests are performed to identify the contaminant. In most cases, positive results require batch rejection, leading to financial losses, production delays, and potential drug shortages.
Best practices for conducting sterility tests
Because sterility testing is qualitative and limited in sensitivity, its accuracy hinges on how the test is performed. Any missteps can produce false positives, casting doubt on the integrity of the entire batch. To ensure that failures reflect true contamination, and not a procedural error, labs must follow rigorous best practices.
Sterility tests should be performed under aseptic conditions, comparable to those used during manufacturing. To verify these conditions, labs should conduct regular environmental and personnel monitoring. As an additional safeguard, barrier technologies like isolators and restricted access barrier systems can physically separate the test from the surrounding environment and personnel.
Only trained and qualified personnel may conduct sterility testing. Training should cover aseptic technique, cleanroom behavior, microbiology, hygiene, gowning, patient safety hazards, and testing procedures. Beyond this, personnel are expected to participate in ongoing training programs and undergo regular evaluations.3
Testing then begins with sample selection. The minimum number of samples is defined in USP <71> based on batch size, but selection must also be representative.1 For aseptically filled products, this means selecting samples from the beginning, middle, and end of the batch, as well as after interventions and excursions. For terminally sterilized products, samples should represent areas that are the most difficult to sterilize. Each run must also include positive and negative controls to confirm test validity.3,4
Throughout the process, complete and accurate documentation is essential. Records should capture environmental data, batch information, and observations during incubation. These records support batch release decisions and guide root cause investigations. After any investigations and on a regular basis, sterility testing procedures should be reviewed and refined. A mindset of continuous improvement supports the reliability of both the testing process and the overall sterility assurance program.
Media fill testing
Sterility testing confirms that the final product is free of viable microorganisms, but it cannot verify the integrity of the aseptic process itself. That’s the role of media fill testing. Also known as aseptic process simulation, media fill testing uses a sterile growth medium in place of the product to assess the potential for a drug unit to become contaminated during normal operations.
The media fill study must closely simulate routine operations, accounting for various aseptic manipulations and interventions, as well as worst-case activities. These studies should also be carried out by trained and qualified personnel.
Both EU GMP Annex 1 and the FDA’s Guidance for Industry outline expectations for media fill testing, including study design, frequency and number of runs, run duration, batch size, media selection, incubation conditions, and interpretation of test results.3,4
Supporting sterile manufacturing
Sterility and media fill tests are essential for ensuring product quality and protecting patient safety. By verifying both the aseptic process and the final product, these methods help maintain compliance with global regulations and instill confidence in every batch released.
However, testing is only as reliable as the practices and tools that support it. That’s why establishing a culture of quality, with rigorous protocols, trained personnel, and carefully selected materials, is just as critical as the tests themselves.
BD understands the complexity and stakes of sterile manufacturing and offers solutions designed to preserve the integrity of the testing environment and enhance efficiency. Its prepared bottled media is available in a variety of formulations, bottle and lid styles, and fill volumes to support diverse operational needs. These bottles can be supplied in EtO packs or double-wrapped configurations, and are manufactured under BD’s ISO 9000-registered Quality Policy, with a sterility assurance level of 10-6.
By focusing on both performance and prevention, and by choosing tools designed with sterility in mind, labs are better equipped to deliver safe, high-quality products batch after batch.
Learn more about BD’s pharmaceutical microbiology solutions.
References
1. United States Pharmacopeia. “<71> Sterility Test.” www.usp.org/harmonization-standards/pdg/general-methods/sterility-test
2. Food and Drug Administration. “Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions.” www.fda.gov/media/73134/download
3. Food and Drug Administration. “Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice.” www.fda.gov/media/71026/download
4. European Commission. “The Rules Governing Medicinal Products in the European Union Volume 4 EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use.” www.gmp-compliance.org/files/guidemgr/20220825_gmp-an1_en_0.pdf
